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1. 发明授权

US08212020B2 Reagents for reversibly terminating primer extension 有权
标题翻译：可逆终止引物延伸的试剂
公开(公告)号：US08212020B2
公开(公告)日：2012-07-03
申请号：US12383306
申请日：2009-03-23
申请人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
发明人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
IPC分类号： C07H17/02 , C07H19/04 , C07H19/10 , C07H19/20 , C07H21/04
CPC分类号： C07H19/10 , C07H19/073 , C07H19/14 , C07H19/173 , C07H23/00
摘要： This invention relates to the field of nucleic acid chemistry, more specifically to the field of compositions of matter that comprise triphosphates of modified 2′-deoxynucleosides and oligonucleotides that are formed when these are appended to the 3′-end of a primer, wherein said modifications comprise NH2 moiety attached to their 3′-hydroxyl group and a fluorescent species in a form of a tag affixed to the nucleobase via a linker that can be cleaved. Such compositions and their associated processes enable and improve the sequencing of oligonucleotides using a strategy of cyclic reversible termination, as outlined in U.S. Pat. No. 6,664,079. Most specifically, the invention concerns compositions of matter that are 5′-triphosphates of ribo- and 2′-deoxyribonucleosides carrying detectable tags and oligonucleotides that might be derived from them. The invention also concerns processes wherein a DNA polymerase, RNA polymerase, or reverse transcriptase synthesizes said oligonucleotides via addition of said triphosphates to a primer.
摘要翻译：本发明涉及核酸化学领域，更具体地涉及包含修饰的2'-脱氧核苷的三磷酸的物质组合物领域和当它们附着于引物的3'-末端时形成的寡核苷酸，其中所述修饰包括连接到它们的3'-羟基上的NH 2部分和通过能够被切割的接头固定在核碱基上的标签形式的荧光物质。这样的组合物及其相关方法使用循环可逆终止的策略实现和改进了寡核苷酸的测序，如美国专利No. 6,664,079。最具体地，本发明涉及携带检测标签的核糖核酸和2'-脱氧核糖核苷的5'-三磷酸的物质组合物，所述可检测标签和寡核苷酸可能来源于它们。本发明还涉及其中DNA聚合酶，RNA聚合酶或逆转录酶通过向引物中加入所述三磷酸合成所述寡核苷酸的方法。

2. 发明授权

US08034923B1 Reagents for reversibly terminating primer extension 有权
标题翻译：可逆终止引物延伸的试剂
公开(公告)号：US08034923B1
公开(公告)日：2011-10-11
申请号：US12383776
申请日：2009-03-27
申请人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
发明人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
IPC分类号： C07H21/02 , C07H21/04 , C07H19/04 , C07H19/048 , C07H19/067 , C07H19/073 , C07H19/167 , C07H19/173
CPC分类号： C07H19/10 , C07H19/20 , C07H21/00
摘要： Processes are disclosed that use 3′-reversibly terminated nucleoside triphosphates to analyze DNA for purposes other than sequencing using cyclic reversible termination. These processes are based on the unexpected ability of terminal transferase to accept these triphosphates as substrates, the unexpected ability of polymerases to add reversibly and irreversibly terminated triphosphates in competition with each other, the development of cleavage conditions to remove the terminating group rapidly, in high yield, and without substantial damage to the terminated oligonucleotide product, and the ability of reversibly terminated primer extension products to capture groups. The presently preferred embodiments of the disclosed processes use a triphosphate having its 3′-OH group blocked as a 3′-ONH2 group, which can be removed in buffered NaNO2 and use variants of Taq DNA polymerase, including one that has a replacement (L616A).
摘要翻译：公开了使用3'-可逆终止的核苷三磷酸以分析DNA作为除了使用循环可逆终止的测序之外的目的的方法。这些过程是基于终端转移酶接受这些三磷酸盐作为底物的意想不到的能力，聚合酶意想不到的能力相互添加可逆地和不可逆地终止的三磷酸酯，发展切割条件以迅速去除终止组，高产生并且对终止的寡核苷酸产物没有实质损害，以及可逆终止的引物延伸产物捕获基团的能力。所公开方法的目前优选的实施方案使用其3'-OH基团被阻断作为3'-ONH2基团的三磷酸酯，其可以在缓冲的NaNO 2中除去，并且使用Taq DNA聚合酶的变体，包括具有替换的变体（L616A ）。

3. 发明申请

US20110275124A1 Reagents for reversibly terminating primer extension 有权
标题翻译：可逆终止引物延伸的试剂
公开(公告)号：US20110275124A1
公开(公告)日：2011-11-10
申请号：US12383306
申请日：2009-03-23
申请人： Steven A. Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
发明人： Steven A. Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
IPC分类号： C12P19/34 , C07H19/14 , C07H19/20 , C07H19/10 , C07H19/073
CPC分类号： C07H19/10 , C07H19/073 , C07H19/14 , C07H19/173 , C07H23/00
摘要： This invention relates to the field of nucleic acid chemistry, more specifically to the field of compositions of matter that comprise triphosphates of modified 2′-deoxynucleosides and oligonucleotides that are formed when these are appended to the 3′-end of a primer, wherein said modifications comprise NH2 moiety attached to their 3′-hydroxyl group and a fluorescent species in a form of a tag affixed to the nucleobase via a linker that can be cleaved. Such compositions and their associated processes enable and improve the sequencing of oligonucleotides using a strategy of cyclic reversible termination, as outlined in U.S. Pat. No. 6,664,079. Most specifically, the invention concerns compositions of matter that are 5′-triphosphates of ribo- and 2′-deoxyribonucleosides carrying detectable tags and oligonucleotides that might be derived from them. The invention also concerns processes wherein a DNA polymerase, RNA polymerase, or reverse transcriptase synthesizes said oligonucleotides via addition of said triphosphates to a primer.
摘要翻译：本发明涉及核酸化学领域，更具体地涉及包含修饰的2'-脱氧核苷的三磷酸的物质组合物领域和当它们附着在引物的3'-末端时形成的寡核苷酸，其中所述修饰包括连接到它们的3'-羟基上的NH 2部分和通过能够被切割的接头固定在核碱基上的标签形式的荧光物质。这样的组合物及其相关方法使用循环可逆终止的策略实现和改进了寡核苷酸的测序，如美国专利No. 6,664,079。最具体地，本发明涉及携带检测标签的核糖核酸和2'-脱氧核糖核苷的5'-三磷酸的物质组合物，所述可检测标签和寡核苷酸可能来源于它们。本发明还涉及其中DNA聚合酶，RNA聚合酶或逆转录酶通过向引物中加入所述三磷酸合成所述寡核苷酸的方法。

4. 发明申请

US20090270601A1 Differential detection of single nucleotide polymorphisms 审中-公开
标题翻译：差异检测单核苷酸多态性
公开(公告)号：US20090270601A1
公开(公告)日：2009-10-29
申请号：US12386595
申请日：2009-04-21
申请人： Steven Albert Benner , Shuichi Hoshika , Nicole Aurora Leal
发明人： Steven Albert Benner , Shuichi Hoshika , Nicole Aurora Leal
IPC分类号： C12P19/34 , C07H21/04
CPC分类号： C12P19/34 , C12Q1/6809 , C12Q2533/101 , C12Q2525/117
摘要： This application claims processes and compositions that enable discovery of single nucleotide polymorphisms (SNPs) and other sequence variation that follows two essentially identical sequences, one a reference, the other a target, as well as SNPs discovered using these processes and compositions. The inventive process comprises preparation of four sets of primers, “T-extendable”, “A-extendable”, “C-extendable”, and “G-extendable”. These primers, when templated on a reference genome, add (respectively) T, A, C, and G to their 3′-ends. The invention also comprises a step where these primer sets are separately bound to complementary sequences on target DNA and, once bound, prime extension reactions using target DNA as the template. If the target DNA directs incorporation of the same nucleotide as the reference DNA, then the T-, A-, C-, and G-extendable primers are extended (respectively) by T, A, C, and G. The architecture of the process distinguishes products from these extensions from products derived if not T, not A, not C and not G (“3N” or “3”, to indicate the other three nucleotides) are not added. Thus, this process discovers differences between the target and reference DNA in the site queried by the primer extension reaction. The distinction makes the two kinds of products either separable or differentially extendable. This distinction is used to disregard products that added T, A, C, and G and to identify the sequence(s) of primers that added not-T, not-A, not-C, and not-G. Further and optionally, information from these sequences identifies loci of the SNPs in an in silico genome.
摘要翻译：本申请要求能够发现单核苷酸多态性（SNP）和遵循两个基本相同的序列的其他序列变异的方法和组合物，一个是参考，另一个靶标，以及使用这些方法和组合物发现的SNP。本发明方法包括制备四组引物“T可延伸”，“可扩展”，“可扩展”和“可扩展”。当在参照基因组上模板时，这些引物将T，A，C和G（分别）添加到它们的3'末端。本发明还包括一个步骤，其中这些引物组分别与靶DNA上的互补序列结合，并且一旦结合，使用靶DNA作为模板进行初始延伸反应。如果目标DNA引导与参考DNA相同的核苷酸引入，则T，A，C和G分别扩增T，A，C和G可延伸的引物。过程将产品从这些扩展区分出来的产品，如果不是T，而不是A，而不是C而不是G（“3N”或“3”，表示其他三个核苷酸）。因此，该过程发现由引物延伸反应查询的位点中的靶标和参照DNA之间的差异。这种区别使得两种产品可分离或差异扩展。这种区别用于忽视添加T，A，C和G的产物，并鉴定未添加-T，而不是A，非-C和非-G的引物序列。进一步和任选地，来自这些序列的信息识别计算机基因组中SNP的位点。

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